US3793153A - Method for propagating yeasts and molds by mixed culturing and method of fermentation thereof - Google Patents

Method for propagating yeasts and molds by mixed culturing and method of fermentation thereof Download PDF

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US3793153A
US3793153A US00147422A US3793153DA US3793153A US 3793153 A US3793153 A US 3793153A US 00147422 A US00147422 A US 00147422A US 3793153D A US3793153D A US 3793153DA US 3793153 A US3793153 A US 3793153A
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acid
culturing
hydrocarbons
lysine
leucine
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Y Miura
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/26—Processes using, or culture media containing, hydrocarbons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi

Definitions

  • the present invention relates to a method for propagating microbial cells wherein yeasts or molds capable of assimilating hydrocarbons or carbohydrates are fermented using hydrocarbons or carbohydrates as a main carbon source, and mixed culturing is carried out by adding bacteria to the fermentation system.
  • the principal considerations in industrial processes are the growth rate of the microorganisms in the culture and the yield of desired metabolic products.
  • the present invention is, in part, based upon the finding that mixed cultures of one or more species of assimilating microorganisms in a culture medium wherein the carbon source is a mixture of numerous hydrocarbons such as petroleum fermentation, the range of assimilated carbon source can be broadened, the rate of carbon conversion can be increased, the kinds of metabolic products can be increased and screening is facilitated.
  • bacteria and molds or yeasts are subjected to mixed culturing in a medium containing hydrocarbons or carbohydrates as a carbon source and together with other nutrient sources necessary for the growth of the microorganisms.
  • the optimum temperature for propragating bacteria is 36C. as compared to 30C. for yeasts and molds, and the division period of yeasts or molds is considerably longer than that of the bacteria
  • the mixed culturing of the present invention has the advantage that the optimum temperature for'propagating yeasts or molds is considerably elevated and approaches that for propagating bacteria.
  • the rate of propagation is also accelerated, the range of assimilated carbon source is remarkably broadened, the yield of cells is increased and the kinds of metabolic products are increased.
  • hydrocarbon and carbohydrate assimilable microorganisms which are useful in the present invention are selected for example, as yeasts, from the families, Cryptococcaceae and Endomycetaceae; as bacteria, from the families Actinomycetaceae, Achromobacteraceae, Bacillaceae, Bacteriaceae, Micrococcaceae, Pseudomonadaceae, Mycobacteriaceae, Brevibacteriaceae and Corynebacteriaceae; and as molds, from the class Ascomycetes.
  • yeasts from the families, Cryptococcaceae and Endomycetaceae
  • bacteria from the families Actinomycetaceae, Achromobacteraceae, Bacillaceae, Bacteriaceae, Micrococcaceae, Pseudomonadaceae, Mycobacteriaceae, Brevibacteriaceae and Corynebacteriaceae
  • molds from the class Ascomycetes.
  • DESCRIPTION Mixed culturing according to the present invention is preferably carried out by fermentation, under aerobic conditions, of an aqueous nutrient media such as by shaking culture or stirring culture. On an industrial scale, it is most advantageous to employ the aerationstirring submerged culture technique.
  • Culturing is generally carried out at a temperature of 25 to 47C. and at a pH OF 3.5 to 8.5. However, when a high production of yeast is desired, it is preferred to adjust the pH to be about 3.5 to 5.5. In the same respect, when a high production of bacteria or mold is desired it is preferred to adjust the pH to be about 6.0 to 8.5. Culturing time is properly determined by the strains of microorganism used, the medium composition and the desired object of culturing. However, the culturing period is generally 8-120 hours.
  • the culture medium in the present invention those having a composition normally used in the ordinary culture of yeasts, molds and bacteria can be used. However, about I to 2 percent by volume of hydrocarbons or carbohydrates based on the total amount of the medium are added thereto as the carbon source. When small amounts of water-soluble vitamins, such as riboflavin, thiamine, p-aminobenzoic acid, pyridoxine hydrochloride, calcium pantothenate, nicotinic acid, biotin, folic acid, etc. are added to the medium, the growth of the microorganisms is increased.
  • water-soluble vitamins such as riboflavin, thiamine, p-aminobenzoic acid, pyridoxine hydrochloride, calcium pantothenate, nicotinic acid, biotin, folic acid, etc.
  • hydrocarbons which can be used as the carbon source gaseous hydrocarbons, naphtha, light oil, kerosene, liquid paraffin, heavy oil, etc. are appropriate. In addition, saturated and unsaturated aliphatic, alicyclic or aromatic compounds may be used.
  • carbohydrates which can be used as the carbon source glucose, glycerol, fructose, sucrose, maltose, mannose, mannitol, xylose, galactose, starch, starch hydrolyzate, molasses, etc. can be used.
  • peptone, NZ amine, meat extract, yeast extract, corn steep liquor, soy bean powders, protein hydrolyzate, inorganic nitrates, ammonium salts, etc. can be used.
  • inorganic salts sodium chloride, calcium chloride, magnesium sulfate, calcium carbonate, phosphates, and small amounts of heavy metal salts such as iron, copper, manganese salts, etc. can be used.
  • hydrocarbons are almost insoluble in water, it is preferable, when added to the liquid medium, to vigorously stir the hydrocarbon with the aqueous solution to form a fine suspension or to add a suitable suspending agent and dissolving agent.
  • two groups of the strains of microorganisms are inoculated in the medium, but these two strains can be precultured separately and then the resulting precultures can be inoculated in the medium at the same time.
  • one of the strains can be inoculated to the other cultured strain and then both can be inoculated in the medium.
  • the two strains of microorganisms to be used may be subjected to mixed culturing in advance and the resulting mixed culture can be inoculated in the main fermentation medium. In any case, the ratio of the amounts of the two strains to be inoculated can be changed as desired according to the purpose of the cul- I ture.
  • the two or more strains are inoculated respectively at a ratio of a microorganism of 10 l cells/cc. and the main culturing is carried out.
  • the strains of the two groups to be inoculated are not restricted to only one kind in the respective groups but more than one strain, that is, any desired number of strains may be included in the respective groups, so long as they can meet the object of the mixed culture.
  • the metabolic end products may be separated from the culture liquid in any well known manner.
  • an amino acid may be separated from the medium after removal of the microorganism cells by means of an ion exchange resin treatment.
  • EXAMPLE I Mycotorula japonica (ATCC 20311) (Strain D) is used as the yeast strain, and Bacillus subtilis (ATCC 21662) (Strain F) is used as the bacterium strain.
  • Each of the strains is initially slant-cultured on potato-agar medium at 27C. for 24 hours.
  • the potatoagar medium comprises 20 percent potato, 3 percent yeast extract and 1 percent hydrocarbon (mixture of l2 26v 1a 2s u so 15 329 and 16 34)- In the case of carbohydrate-assimilable microorganisms, 1 percent glucose is used in place of the hydrocarbons.
  • thestrains are respectively inoculated by one platinum loop into 500 cc. Erlenmyer flasks containing a basal medium comprising 10 ml./l. hydrocarbons (mixture of n-C I-I n-C H n-C l-l n-c, H,, and n-C I-I 5.0 g./l. NI-l I-I PO 4.0 g./l. KH PO. 0.3 g./l. K d-IP0 0.4 g./l. MgSO -7I-I O, 0.1 g./l. NaCl, 0.1 g./l.
  • the above cultures are then precultured at a temper ature of 30C. withrespect to the yeast and, 46C. with respect to the bacterium for'48 hours with shaking at an amplitude of 5 cm. and 100 reciprocations per minute.
  • the thus obtained precultures are respectively centrifuged at 3,000 revolutions per minute for 10 minutes to collectthe microbial cells which are thereafter washed with a 0.1 M'phosphate bufier solution (pH 7.0).
  • the washed cells are then respectively inoculated into a basal medium of the same composition as given above in such manner so that the cultures contain 10 10" cells/cc. Fermentation of the main culture is carried out with shaking and thereafter the cells are counted under a microscope with a Thomas blood cell counter for the yeast strain employed and with a bacteria counter chamber for the bacterium strain employed. For the mixed culture, the number of respective microbial cellsare counted under a microscope in M oldsi K: Periicilliurh jahthihel idrh TIFIGWEVZAICC In FIG. 1A, the bacterium strain A and yeast strainoare cultivated separately. After 48 hours, both strains reach "maximum cell growth. However, as shown in FIG. 13, maximum cell concentration occurs at about 38 hours with both strains plateauing at about the same time.
  • FIG. 2A shows the results of mixed culturing at 42C.. As is evident from the FIG. 2B, both strains exhibit maximum propagation after only about 16 hours of cultivation. In addition, both strains propagate to a much greater extent than in single culturing.
  • H Pseudomonas aeruginosa I.F.O. 3923 (ATCC 21661) 7
  • I Corynebacterium hydrocarboclastus (ATCC 6946)
  • J Brevibacterium acetylicum I.F.O. 12146 (ATCC Cultivatioifis cam" "ea an? iii mesasraarasea in Example I except that the cultivation temperature is varied and the carbon source is changed in accordance with the microorganisms used.
  • the variables, percent- 12552 (ATCC ages of yields of the cells and the maximum specific microbial cells are shown in the following Tables I and 'growth rates calculate on the basis of the numbers of II:
  • Citric Acid HI- m K I 37C. n-alkanes of 937 K 0.29l
  • 0.289 Phenylalanine, Polycarboxylic acids, Citric C5-C1" acid,Glutamic acid, Alanine, Valine,
  • Yeasts 77 came 'zro'pibaiis (Y 2) Candida intermedia (Y 3 Candida lipolytica (Y 4) Candida albicans (Y 5) Candida rugosa (Y 6) Candida petrophilum (Y 7) Candida brumptii (Y 8) Candida catenulata (Y 9) Candida melinii (Y l) Candida parapsilasis (Y 11) Candida pulcherrima (Y 12) Candida reuêtii (Y 13) Candida cloacae TABLE 3 -Continued Single culturing (ycust) (carbon sources: hydrocarbons) (Y 14) Candida maltosa (Y 15) Candida tenuis (Y 16) Saccharomyceteae pichia guilliermondii (Y 17) Saccharomyceteae pichia farinasa (Y 18) Saccharomyceteae pichia vini (Y l9) Saccharomyceteae debaryo

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US00147422A 1970-06-06 1971-05-27 Method for propagating yeasts and molds by mixed culturing and method of fermentation thereof Expired - Lifetime US3793153A (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4013508A (en) * 1974-11-21 1977-03-22 Liquichimica S.P.A. Process for the production of 1-aspartic acid by fermentation of hydrocarbons
US4220720A (en) * 1977-12-14 1980-09-02 Bio Research Center Co., Ltd. Manufacture of fatty acids having straight and long carbon chains using a microorganism
US4302542A (en) * 1976-06-21 1981-11-24 Phillips Petroleum Co. Fermentation with thermophilic mixed cultures
US4311511A (en) * 1976-07-07 1982-01-19 Gernot Graefe Method for producing high-grade fertilizer
US4439523A (en) * 1981-07-07 1984-03-27 Phillips Petroleum Company Production of single cell protein material
US20050281895A1 (en) * 1999-11-05 2005-12-22 Sanfoi-Synthelabo Slimming cosmetic composition containing a substance inducing the production of IL-6

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2704866B1 (fr) * 1993-05-06 1995-07-21 Elf Antar France Milieu de culture d'une flore microbienne procédé de préparation d'un inoculum lyophilisé et inoculum obtenu par ce procédé.
CN113913309B (zh) * 2021-09-17 2023-03-31 中国科学院成都生物研究所 一株耐碱酵母及在利用沼液产单细胞蛋白中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1875401A (en) * 1932-09-06 of tebre haute
US2636823A (en) * 1947-11-10 1953-04-28 Schenley Ind Inc Method of making biosynthesized product
US2766176A (en) * 1953-02-11 1956-10-09 George A Jeffreys Process for culturing anaerobic bacteria
US3536586A (en) * 1968-01-25 1970-10-27 Squibb & Sons Inc Microbiological process for simultaneously 1-dehydrogenating and 16-hydroxylating a steroid
US3667968A (en) * 1969-05-13 1972-06-06 Beatrice Foods Co Cheese flavors

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1875401A (en) * 1932-09-06 of tebre haute
US2636823A (en) * 1947-11-10 1953-04-28 Schenley Ind Inc Method of making biosynthesized product
US2766176A (en) * 1953-02-11 1956-10-09 George A Jeffreys Process for culturing anaerobic bacteria
US3536586A (en) * 1968-01-25 1970-10-27 Squibb & Sons Inc Microbiological process for simultaneously 1-dehydrogenating and 16-hydroxylating a steroid
US3667968A (en) * 1969-05-13 1972-06-06 Beatrice Foods Co Cheese flavors

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4013508A (en) * 1974-11-21 1977-03-22 Liquichimica S.P.A. Process for the production of 1-aspartic acid by fermentation of hydrocarbons
US4302542A (en) * 1976-06-21 1981-11-24 Phillips Petroleum Co. Fermentation with thermophilic mixed cultures
US4311511A (en) * 1976-07-07 1982-01-19 Gernot Graefe Method for producing high-grade fertilizer
US4311510A (en) * 1976-07-07 1982-01-19 Gernot Graefe Method for producing high-grade fertilizer
US4220720A (en) * 1977-12-14 1980-09-02 Bio Research Center Co., Ltd. Manufacture of fatty acids having straight and long carbon chains using a microorganism
US4439523A (en) * 1981-07-07 1984-03-27 Phillips Petroleum Company Production of single cell protein material
US20050281895A1 (en) * 1999-11-05 2005-12-22 Sanfoi-Synthelabo Slimming cosmetic composition containing a substance inducing the production of IL-6
US20090123455A1 (en) * 1999-11-05 2009-05-14 Sanofi-Aventis Slimming cosmetic composition containing a substance inducing the production of il-6
US8628770B2 (en) * 1999-11-05 2014-01-14 Sanofi Slimming cosmetic composition containing a substance inducing the production of IL-6

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FR2109603A5 (enExample) 1972-05-26

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